Project 2: Subtype Specification and Arrhythmogenic Potential of Stem Cell Derived Cardiomyocytes. Cardiomyocytes derived from human embryonic stem cells (hESCs) have tremendous promise for cardiac repair, both for replacing ventricular myocardium after an infarct and serving as a biologic pacemaker in diseases like sick-sinus syndrome. We have reported methods to generate large quantities of highly purified cardiomyocytes from hESCs, but these preparations include cells with distinct nodal/pacemaker and working (i.e. chamber-like) phenotypes. For these cells to be useful and safe, they must have electrophysiological properties matched to each application. Hence, this project will develop approaches to control the cardiac subtype of hESC derivatives and then will determine their electrophysiological behavior following transplantation in intact and infarcted hearts. In Aim 1, we will develop a genetic selection system to follow the lineage relationships of cardiac subtypes derived from hESCs. This work builds on our preliminary findings that a promoter element from the cGATA6 gene identifies hESC-derived cardiomyocytes with the nodal phenotype. In Aim 2, we will elucidate the molecular events by which neuregulin/ErbB signaling regulates the cardiac subtype of hESC-derived cardion\yocytes. This work will employ the genetic reporters from Aim 1 but will also lead to complementary pharmacological approaches to control cardiac subtype specification. In Aim 3, we will address two uncertainties regarding the electrophysiological behavior of hESC-derived cardiomyocytes following transplantation. First, we will test whether these cells are electrically coupled and beat synchronously with host muscle in normal and infarcted hearts. Second, we will test the hypothesis that their transplantation will modulate the incidence of infarct-related arrhythmias, while the incidence of graft-associated arrhythmias can be tuned by controlling the phenotype of the input cell preparation.